FGF2 regulates otic gene expression through the MAP kinase pathway.

<p>(A): HH stage 8 embryos were cultured for 18 hours in the presence of different FGF pathway inhibitors and assayed for expression of Pax2. Only embryos treated with the FGFR inhibitor SU5402 or the MEK kinase inhibitor U0126 showed decreased or abolished Pax2 expression. (B): FGF signaling is present in presumptive otic placodal ectoderm prior to the onset of otic placode genes. HH stage 6 and 7 embryos were either stained with antibodies to phosphorylated ERK kinase or processed for in situ hybridization with the FGF target gene PEA3. Electroporation of a dominant-negative MEK construct (together with a GFP reporter) down-regulates Pax2 expression in the otic region (asterisks) but not in control embryos (yellow arrow) or on the contralateral side of the embryo (arrowheads). (C): Inhibition of the MAP kinase pathway also blocks induction of Pax2 in cultured chunks of the developing otic region or in FGF-treated trigeminal level ectoderm. Embryo chunks or ectoderm pieces were sectioned and stained with antibodies to Pax2. The MEK inhibitor U0126 significantly reduced induction of Pax2 at both concentrations tested. (D): The MEK kinase inhibitor U0126 can also block the induction of other otic genes in addition to Pax2. Trigeminal level ectoderm was cultured with 50 ng/ml FGF2 in the presence or absence of 20 µM U0126. QPCR for the otic genes <i>Spry2, EphA4</i> and <i>Elk3</i> were all significantly reduced by U0126 treatment (p<0.05 in each case).